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Total RNA was extracted from tissues. The Iso-Seq library was prepared according to the Isoform Sequencing protocol (Iso-Seq) using the ClontechSMARTer PCR cDNA Synthesis Kit (Clontech: 634925 or 634926) and the BluePippin Size Selection System protocols described by Pacific Biosciences with the following modifications. All of our Iso-Seq libraries were prepared from polyA+ RNA purified from total RNA from organisms. For cDNA conversion, 3 ug of total RNA was used as input into the ClontechSMARTer reaction using SMARTer PCR cDNA Synthesis kit (Clontech: 634925 or 634926). The cDNA library was constructed using the ClontechSMARTer PCR cDNA Synthesis Kit and the KAPAHIFI PCR kit (Pacific Biosciences, Inc.). The fragment was screened using the BluePippin Size Selection System (Sage Science, Inc.) and the libraries were constructed using the SMRTbellTM Template prep kit (Pacific Biosciences, Inc.). Libraries were prepared for sequencing by annealing a sequencing primer and binding polymerase (Pacific Biosciences, Inc.) to the primer annealed template. The polymerase-bound template was bound to MagBeads (P/N 100-125-900). After the library were built, sequencing was performed on PacBio RS II platform with the latest sequencing reagents DNA Binding kit P6 (Pacific Biosciences, Inc.) and DNA Sequencing Kit 4.2 (Pacific Biosciences, Inc.). |
ISO-Seq |
TRANSCRIPTOMIC |
other |
SINGLE
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